High Epidermal Growth Factor Receptor Research Articles

Selective regain of egfr gene copies in CD44+/CD24-/lowbreast cancer cellular model MDA-MB-468

BackgroundIncreased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies.MethodsThe basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique.ResultsLow and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies.ConclusionProgressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.

Nuclear EGFR in ductal invasive breast cancer: correlation with cyclin-D1 and prognosis

The epidermal growth factor receptor (EGFR)-family and cyclin-D1 have been extensively studied in breast cancer; however systematic studies that examine protein expression and gene status in the same cohort of patients are lacking. Also emerging evidences suggest existence of a direct EGFR-signaling pathway, which involves cellular transport of EGFR from cell membrane to the nucleus, and transcriptional regulation of the target genes. Thus, we examined the protein expression of membrane EGFR, nuclear EGFR, cyclin-D1 and the corresponding gene status in 113 breast carcinomas by immunohistochemistry and fluorescence in situ hybridization using tissue microarrays. Membrane EGFR overexpression and EGFR gene amplification were detected in 2% cases, while nuclear EGFR was detected in 40% of cases, with 12% having high nuclear EGFR staining. Nuclear EGFR correlated with tumor size (P=0.0005), lymph node metastasis (P=0.0288), Nottingham prognostic index (P=0.0011) and estrogen receptor (ER) expression (P=0.0258) but the letter correlation was observed only in premenopausal group of patients. Strong cyclin-D1 expression and cyclin-D1 gene (CCND1) amplification were found in 64 and 13% of the cases, respectively. Cyclin-D1 expression showed positive correlation with ER (P=0.0113) and inverse correlation with Nottingham prognostic index (P=0.0309) and membrane EGFR (P=0.0201). CCND1 amplification also showed inverse correlation with membrane EGFR (P=0.0420). A strong correlation between membrane EGFR expression and gene amplification (P=0.0035), as well as cyclin-D1 overexpression and gene amplification (P=0.0362), was demonstrated. On univariate analysis cyclin-D1 expression showed a correlation with longer overall survival in the premenopausal group and nuclear EGFR correlated with shorter overall survival in whole cohort as well in the premenopausal group of patients. Multivariate analysis revealed nuclear EGFR to be an independent prognostic factor and showed 3.4 times greater mortality risk for nuclear EGFR+++ patients as compared with nuclear EGFR negative patients (hazard ratio =3.402; P=0.0026).

MRI-coupled Fluorescence Tomography Quantifies EGFR Activity in Brain Tumors

This report demonstrates the diagnostic potential of magnetic resonance imaging (MRI)-coupled fluorescence molecular tomography (FMT) to determine epidermal growth factor receptor (EGFR) status in brain cancer. Two orthotopic glioma xenograft models were used in this study: one represented high EGFR expression and the other low expression. Nude mice were inoculated with cells from either one of the tumor lines or were used in a sham surgery control group. Animals were imaged using a unique MRI-FMT scanner 48 hours after intravenous injection of a near-infrared fluorophore bound to epidermal growth factor (EGF) ligand. Coronal images of fluorescence activity of the injected dye in the mouse brain were recovered using the MRI images as anatomical templates. In vivo images of fluorescence activity showed significant differences between animal populations, an observation confirmed by receiver operating characteristic analysis that revealed 100% sensitivity and specificity between animal groups implanted with EGFR((+)) and EGFR((-)) tumor lines. Similar performance was observed between EGFR((+)) and sham surgery control animals. This preclinical study suggests that MRI-FMT with fluorescent EGF provides excellent discrimination between tumors based on EGFR status. Reliable quantification of receptor status using minimally invasive techniques would be an important innovation for investigating new and existing cancer treatments that target these cellular mechanisms in research animals, and may be applied to identify receptor amplification in human brain cancer patients. This study represents the first systematic multianimal validation of receptor-specific imaging using MRI-guided fluorescence tomography.

Activating KRAS Mutations and Overexpression of Epidermal Growth Factor Receptor as Independent Predictors in Metastatic Colorectal Cancer Patients Treated With Cetuximab

Cetuximab, a monoclonal antibody targeting epidermal growth factor receptor (EGFR), has been proven to be efficient in metastatic colorectal cancer (mCRC); however, the therapeutic response is variable and markers predictive of response are urgently required. This study was conducted to determinate the predictive values of KRAS mutation status and EGFR expression in mCRC patients treated with cetuximab plus chemotherapy. Clinical benefit with EGFR-targeting antibodies seems to be restricted to a particular subgroup of mCRC patients. Therefore, the identification of reliable predictive factors for mCRC patients is imperative before the introduction of targeted chemotherapy. Ninety-five mCRC patients receiving cetuximab plus the FOLFIRI or FOLFOX-4 chemotherapy were enrolled into the present study. KRAS mutation status/EGFR expression levels were analyzed using direct sequencing, immunohistochemistry (IHC), and reverse transcription-polymerase chain reaction (RT-PCR) assay, respectively. The association between clinical response, progression-free survival (PFS) and overall survival (OS) as well as KRAS mutation status/EGFR expression levels were evaluated. Of 95 mCRC patients, KRAS mutations were identified in 41 cases, and EGFR overexpression (protein or mRNA levels) were observed in 78 patients. Among 41 tumors with KRAS mutation, 33 were found to be activating mutants at codons 12, 13, 15 or 18, while 8 were nonactivating mutants at codons 20, 30, or 31. Fifty-five patients responded to cetuximab plus chemotherapy, 49 were EGFR overexpression and 46 were wild-type KRAS tumor status. Patients with tumors that express high EGFR levels or harbor wild-type KRAS are more likely to have a better PFS and OS when treated with cetuximab plus chemotherapy (all P < 0.05). Furthermore, patients with nonactivating KRAS mutants in tumors had a significantly better PFS and OS than patients with activating KRAS mutants (both P < 0.05). However, for patients with wild-type KRAS tumor status, EGFR expression remains a relevant predictor of clinical response. The study suggests that activating KRAS mutants is a particularly important independent predictive marker in mCRC patients treated with cetuximab plus chemotherapy, of which combing activating KRAS mutants and EGFR could help to identify the subgroup of patients who are most likely to respond to cetuximab plus chemotherapy.

Molecular Predictors of Outcome With Gefitinib and Docetaxel in Previously Treated Non–Small-Cell Lung Cancer: Data From the Randomized Phase III INTEREST Trial

PURPOSE In the phase III INTEREST trial, 1,466 pretreated patients with advanced non-small cell lung cancer (NSCLC) were randomly assigned to receive gefitinib or docetaxel. As a preplanned analysis, we prospectively analyzed available tumor biopsies to investigate the relationship between biomarkers and clinical outcomes. METHODS Biomarkers included epidermal growth factor receptor (EGFR) copy number by fluorescent in situ hybridization (374 assessable samples), EGFR protein expression by immunohistochemistry (n = 380), and EGFR (n = 297) and KRAS (n = 275) mutations. Results For all biomarker subgroups analyzed, survival was similar for gefitinib and docetaxel, with no statistically significant differences between treatments and no significant treatment by biomarker status interaction tests. EGFR mutation-positive patients had longer progression-free survival (PFS; hazard ratio [HR], 0.16; 95% CI, 0.05 to 0.49; P = .001) and higher objective response rate (ORR; 42.1% v 21.1%; P = .04), and patients with high EGFR copy number had higher ORR (13.0% v 7.4%; P = .04) with gefitinib versus docetaxel. CONCLUSION These biomarkers do not appear to be predictive factors for differential survival between gefitinib and docetaxel in this setting of previously treated patients; however, subsequent treatments may have influenced the survival results. For secondary end points of PFS and ORR, some advantages for gefitinib over docetaxel were seen in EGFR mutation-positive and high EGFR copy number patients. There was no statistically significant difference between gefitinib and docetaxel in biomarker-negative patients. This suggests gefitinib can provide similar overall survival to docetaxel in patients across a broad range of clinical subgroups and that EGFR biomarkers such as mutation status may additionally identify which patients are likely to gain greatest PFS and ORR benefit from gefitinib.

Abstract C49: Combination treatment with metronomic cyclophosphamide and the EGFR monoclonal antibody nimotuzumab is efficacious and non-toxic in a preclinical model of advanced triple negative breast cancer

Abstract Low dose, metronomic chemotherapy with cyclophosphamide has shown activity in the treatment of advanced breast cancer, and has been paired with targeted agents such as trastuzumab and bevacizumab for long term therapy, with encouraging results. The epidermal growth factor receptor (EGFR) pathway may be a relevant target for the treatment of triple-negative breast cancer, a tumor type with an aggressive behavior and limited therapeutic options. Nimotuzumab is a humanized monoclonal antibody targeting EGFR with an affinity that is lower than currently approved agents, such as cetuximab and panitumumab. This leads to preferential targeting of EGFR-overexpressing tumors, minimizing toxicity, making it well suited for long term dosing, including possible combination therapy with minimally toxic chemotherapeutic regimens, when treating tumors with high EGFR expression. The objective of this study was to evaluate nimotuzumab in combination with metronomic cyclophosphamide in a preclinical model of advanced, triple negative breast cancer. Increasingly aggressive metastatic variants of the MDA-MB-231 human breast cancer cell line were selected through passage in vivo, resulting in a line termed 164/8-1B, characterized as highly metastatic to many organs, including lymph nodes, lung, and brain. This variant also had increased EGFR expression compared to the parental cell line. Combination therapy with nimotuzimab twice weekly and continous low-dose cyclophosphamide resulted in significant primary tumor growth delay in the metastatic variant, compared to the parental cell line. In addition, the combination resulted in a significant survival advantage over cyclophosphamide alone in the advanced metastatic model. Nimotuzumab displayed single agent activity in primary tumor xenografts but not in the treatment of advanced metastatic disease. Primary tumor regrowth in the mammary fat pad occurred with similar frequency across treatment groups, however the resulting tumor size was decreased in mice receiving combination treatment. Lymph node metastasis was observed in 40–60% of mice in each treatment group, with the exception of those that received combination therapy, where no gross evidence of lymphadenomegaly was observed. Finally, cell lines established from lung metastases from 3 mice receiving combination therapy showed EGFR expression, suggesting that resistance to treatment was not associated with loss of target expression. In conclusion, combination therapy with low dose, continuous cyclophosphamide and the EGFR monoclonal antibody nimotuzumab was an efficacious regimen in this advanced, metastatic, triple negative, EGFR overexpressing breast cancer model, and would be expected to be minimally toxic to patients. This combination approach should be considered for this patient population in future clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C49.

Abstract B221: Expression of EGFR ligands in non-small cell lung carcinoma xenografts as potential biomarkers for classical or targeted therapies

Abstract Increased levels of the epidermal growth factor receptor (EGFR) and its cognate ligands are common in non-small cell lung cancer (NSCLC) and correlate with a poor prognosis. In the last years targeted therapies against the EGFR were developed, but results of clinical trials were rather disappointing. The identification of biomarkers in NSCLC as predictive factors for an individualized therapy is urgently needed. Twenty six xenografts were established from fresh surgical material from NSCLC patients and characterized with regard to their sensitivity to four cytostatics (Carboplatin, Etoposide, Paclitaxel, Gemcitabine) and two EGFR-inhibitors (Cetuximab, Erlotinib). The expression of EGFR and its ligands epidermal growth factor (EGF), transforming growth factor alpha (TGFα), epiregulin and amphiregulin were examined at mRNA and protein level. Three ligands, which bind exclusively to EGFR (EGF, TGFα and amphiregulin) showed correlations to the mRNA expression of the receptor. Also the mRNA-levels of amphiregulin and epiregulin correlated with each other. The responsiveness towards Etoposide correlated negatively with the EGF-protein (r=-0.479, P&amp;lt;0.05) and with the EGFR-mRNA (r=-0.663, P&amp;lt;0.01) expression. This could mean that the expression of the EGFR-pathway related molecules is able to protect tumor cells from the induction of apoptosis through a cytostatic drug. The response rate to Gemcitabine showed a negative correlation with the TGFα-mRNA level (r=-0.525, P&amp;lt;0.01). Controversially, the amphiregulin-mRNA level correlated positively with the responsiveness to Gemcitabine (r=0.553, P&amp;lt;0.01). A high level of TGFα-mRNA correlated with the resistance to Erlotinib (r=-0.456, P&amp;lt;0.05). The epiregulin-mRNA level showed a positive correlation concerning the sensitivity towards Cetuximab (r=0.476, P&amp;lt;0.05). High EGFR levels seem to be indicative for the resistance to Erlotinib (r=0.636, P&amp;lt;0.05) in squamous cell carcinomas and for the sensitivity to Cetuximab (r=-0.888, P&amp;lt;0.05) in adenocarcinomas. The results of the present study showed the role of the EGFR activated pathways in the dynamics of tumor progression in NSCLC on the one hand and reflect the high level of redundancy and interrelationships of the EGFR signalling pathway on the other hand. The patient-derived NSCLC models are suitable tools for the identification of biomarkers and their regulation mechanisms in a clinically related and standardized way. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B221.

Cross-Talk between Estrogen Receptor and Epidermal Growth Factor Receptor in Head and Neck Squamous Cell Carcinoma

This study aimed to characterize estrogen receptor expression and signaling in head and neck squamous cell carcinoma (HNSCC) cell lines and patient tissues, and to evaluate estrogen receptor and epidermal growth factor (EGF) receptor (EGFR) cross-activation in HNSCC. Estrogen receptor expression and signaling in HNSCC cell lines were assessed by immunoblotting. In vitro proliferation and invasion were evaluated in HNSCC cell lines in response to estrogen receptor and EGFR ligands or inhibitors. Estrogen receptor and EGFR protein expression in patient tissues was assessed by immunohistochemical staining. Phospho-mitogen-activated protein kinase (P-MAPK) levels were significantly increased following combined estrogen and EGF treatment. Treatment of HNSCC cells with estrogen and EGF significantly increased cell invasion compared with either treatment alone, whereas inhibiting these two pathways resulted in reduced invasion compared with inhibiting either pathway alone. EGFR (P = 0.008) and nuclear estrogen receptor alpha (ER alpha(nuc); P < 0.001) levels were significantly increased in HNSCC tumors (n = 56) compared with adjacent mucosa (n = 30), whereas nuclear estrogen receptor beta (ER beta(nuc)) levels did not differ (P = 0.67). Patients with high ER alpha(nuc) and EGFR tumor levels had significantly reduced progression-free survival compared with patients with low tumor ER alpha(nuc) and EGFR levels (hazards ratio, 4.09; P = 0.01; Cox proportional hazards). In contrast, high ER beta(nuc) tumor levels were not associated with reduced progression-free survival alone or when combined with EGFR. ER alpha and ER beta were expressed in HNSCC, and stimulation with estrogen receptor ligands resulted in both cytoplasmic signal transduction and transcriptional activation. Estrogen receptor and EGFR cross-talk was observed. Collectively, these studies indicate that estrogen receptor and EGFR together may contribute to HNSCC development and disease progression.

Somatic Mutation of Epidermal Growth Factor Receptor in a Small Subset of Cutaneous Squamous Cell Carcinoma

Somatic Mutation of Epidermal Growth Factor Receptor in a Small Subset of Cutaneous Squamous Cell Carcinoma

Detecting Epidermal Growth Factor Receptor Tumor Activity In Vivo During Cetuximab Therapy of Murine Gliomas

Noninvasive molecular imaging of glioma tumor receptor activity was assessed with diagnostic in vivo fluorescence monitoring during targeted therapy. The study goals were to assess the range of use for treatment monitoring and stratification of tumor types using epidermal growth factor (EGF) receptor (EGFR) status with administration of fluorescently labeled EGF and determine its utility for tumor detection compared to magnetic resonance imaging (MRI). EGFR+ and EGFR- glioma tumor lines (human glioma [U251-GFP] and rat gliosarcoma [9L-GFP], respectively) were used to assess these goals, having a 20-fold difference between their EGF uptakes. Treatment with cetuximab in the EGFR+ tumor-bearing animals led to decreased EGF tumor uptake, whereas for the EGFR- tumors, no change in fluorescence signal followed treatment. This diagnostic difference in EGFR expression could be used to stratify the tumor-bearing animals into groups of potential responders and nonresponders, and receiver-operating characteristic curve analysis revealed an area under the curve (AUC) of 0.92 in separating these tumors. The nonlocalized growth pattern of U251-GFP tumors resulted in detection difficulty on standard MRI, but high EGFR expression made them detectable by fluorescence imaging (AUC = 1.0). The EGFR+ U251-GFP tumor-bearing animals could be noninvasively stratified into treated and untreated groups on the basis of fluorescence intensity difference (P = .035, AUC = 0.90). EGFR expression was tracked in vivo with fluorescence and determined to be of use for the stratification of EGFR+ and EGFR- tumors, the detection of EGFR+ tumors, and monitoring of molecular therapy.

Comparison of FISH, PCR, and Immunohistochemistry in Assessing EGFR Status in Lung Adenocarcinoma and Correlation With Clinicopathologic Features

The purpose of this study is to investigate the relationship between mutation status, copy number, and protein expression of epidermal growth factor receptor (EGFR) in lung adenocarcinoma, and to correlate the genetic status and clinicopathologic features. Forty-nine adenocarcinomas were tested by polymerase chain reaction (PCR) for EGFR gene mutations, by fluorescence in situ hybridization (FISH) for increased EGFR gene copy number, and by immunohistochemistry (IHC) for EGFR protein expression. Terminal respiratory unit (TRU) morphology, tumor grade, mitotic count, and pleural and lymphovascular invasion were also examined. Five of 49 tumors (10%) had EGFR mutation by PCR. Eighteen (36%) had high EGFR gene copy number by FISH, all of which were polysomies without gene amplification. Fifteen (31%) had EGFR protein overexpression by IHC, 13 (87%) of which were also positive by FISH. Of the PCR-positive cases, 4/5 (80%) were positive by FISH and 3/5 (60%) were positive by IHC. TRU morphology was observed in all PCR-positive cases and in about 50% of FISH-positive and IHC-positive cases. Pleural invasion was significantly more common in IHC and FISH-positive cases, and lymphovascular invasion was more often present in FISH-positive cases. No statistically significant differences in mitotic count, age, sex, smoking status, degree of differentiation, or stage were seen. Our findings suggest that a staged approach, with FISH testing as the first step, followed by PCR for the FISH-negative cases would be most effective in the identification of patients with EGFR gene alterations.

Comparison of automated silver enhanced in situ hybridization and fluorescence in situ hybridization for evaluation of epidermal growth factor receptor status in human glioblastomas

The epidermal growth factor receptor (EGFR) is amplified in approximately 40% of glioblastomas making it a compelling molecular target for therapy. Before starting a therapy targeting the EGFR pathway, accurate determining of EGFR status is a prerequisite. We evaluated the reliability of the novel automated silver enhanced in situ hybridization for the detection of EGFR gene amplification in human glioblastomas. EGFR-amplification status was assessed in 93 cases of glioblastoma by silver enhanced in situ hybridization and compared with results of fluorescence in situ hybridization and immunohistochemistry. In a second cohort, silver enhanced in situ hybridization status was correlated with EGFR gene expression data. The EGFR gene was amplified in 25/90 tumours (28%) by silver enhanced in situ hybridization, and in 28/93 tumours (30%) by fluorescence in situ hybridization. The concordance rate for silver enhanced in situ hybridization and fluorescence in situ hybridization was 98%. Two glioblastomas were scored as being amplified by fluorescence in situ hybridization but not by silver enhanced in situ hybridization. Polymerase chain reaction-based EGFR-amplification data were highly correlated with EGFR silver enhanced in situ hybridization. Altogether, 81 of 91 cases (89%) showed positivity for EGFR expression by immunohistochemistry. Although EGFR protein over expression was associated with gene amplification (r=0.40, P<0.001), there were 29 of 91 cases that showed a high EGFR protein level and no EGFR amplification by fluorescence in situ hybridization. The high concordance rate of silver enhanced in situ hybridization and fluorescence in situ hybridization for the detection of EGFR amplification in paraffin-embedded glioblastomas samples demonstrates that silver enhanced in situ hybridization is a valid and attractive alternative to fluorescence in situ hybridization. Silver enhanced in situ hybridization combines the advantages of bright field microscopy with fully automated analysis in a cost-effective way thereby emphasizing its use for routine application in surgical pathology.

Protein overexpression and gene amplification of epidermal growth factor receptor in nonsmall cell lung carcinomas: Comparison of four commercially available antibodies by immunohistochemistry and fluorescence in situ hybridization study

Epidermal growth factor receptor (EGFR) overexpression in nonsmall cell lung carcinomas (NSCLC) is variable ranging from 19% to 89% and its prognostic value remains controversial. We tried to investigate (1) EGFR protein expression using four different antibodies, (2) the correlation between protein overexpression and EGFR gene amplification, and (3) the correlation between EGFR genetic status and clinicopathologic features in NSCLC. We examined EGFR protein expression using four different antibodies including Zymed EGFR kit (Clone 31G7), Dako EGFR pharmDx kit (Clone 2-18C9), Dako (Clone H11) and Novocastra (Clone EGFR 113) by immunohistochemical analysis. The protein overexpression was compared to gene amplification status by fluorescence in situ hybridization (FISH). EGFR protein overexpression was observed in 56% of tumors with Zymed EGFR kit, in 51% with Dako EGFR pharmDx kit, in 5% with Dako and in 18% with Novocastra ( p = 0.010). Both Zymed and Dako pharmDx kit were more sensitive than the Dako test (clone H11) and Novocastra clone EGFR 113. EGFR overexpression was more prominent in squamous cell carcinomas (SCC) than adenocarcinomas (ADC) (71% vs. 48%, p = 0.001 with Zymed, 61% vs. 45%, p = 0.011 with Dako pharmDx kit; respectively). EGFR FISH-positivity as represented by high polysomy and gene amplification was observed in 45% of the NSCLC patients. Protein expression levels significantly correlated with the gene copy number per tumor cell ( p < 0.001). Our data showed a higher percentage of positive cells detected by Zymed and Dako pharmDx tests. The EGFR protein overexpression rate varied from 4% to 72% according to different antibody clones and histologic types. EGFR protein expression detected by Zymed and Dako pharmDx was significantly associated with a high EGFR gene copy number.

PET of EGFR Antibody Distribution in Head and Neck Squamous Cell Carcinoma Models

Epidermal growth factor receptor (EGFR) is a well-characterized protooncogene that has been shown to promote tumor progression in solid cancers. Clinical results for EGFR targeting with specific monoclonal antibodies (mAbs) such as cetuximab and panitumumab are promising; however, most studies indicate that only a subgroup of patients receiving the mAbs benefit from the immunotherapy, independent of EGFR expression level. To understand the in vivo kinetics of antibody delivery and localization, we performed small-animal PET studies with (64)Cu-labeled panitumumab in xenografts derived from 3 cell lines of human head and neck squamous cell carcinoma (HNSCC). Nude mice bearing HNSCC tumors with different levels of EGFR expression were imaged with small-animal PET using (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-panitumumab. Antibody distribution in the tumors was confirmed by ex vivo immunostaining using panitumumab and fluorescein 5(6)-isothiocyanate (FITC) panitumumab. CD31 immunostaining and Evans blue assay were also performed to assess the tumor vascular density and permeability. Among these 3 tumor models, UM-SCC-22B tumors with the lowest EGFR protein expression showed the highest (64)Cu-DOTA-panitumumab accumulation, whereas SQB20 tumors with the highest EGFR expression showed the lowest (64)Cu-DOTA-panitumumab accumulation. Ex vivo staining demonstrated that SQB20 cells still had extremely high EGFR expression after forming tumors in nude mice, indicating that the low uptake of (64)Cu-DOTA-panitumumab in SQB20 tumors was not due to the loss of EGFR expression. The results from CD31 immunostaining and Evans blue permeability assay suggest that the low vessel density, poor vascular permeability, and binding site barrier are likely responsible for the overall low tumor uptake of the highly EGFR-expressing SQB20 tumors. The results from this study provide a possible explanation for the lack of an observed correlation between therapeutic efficacy of cetuximab and panitumumab and EGFR expression level as determined by immunohistochemistry or fluorescent in situ hybridization and may shed new light on the complications of anti-EGFR mAb therapy for HNSCC and other malignancies.

High Epidermal Growth Factor Receptor Expression in Metastatic Colorectal Cancer Lymph Nodes May Be More Prognostic of Poor Survival Than in Primary Tumor

High epidermal growth factor receptor (EGFR) expression in the primary tumor predicts poor survival in colorectal cancer. However, EGFR expression may be discordant in primary tumor versus nodal or distant metastasis. We evaluated prognostic values of high EGFR expression in nodal metastasis versus primary tumor tissue. Tissue microarrays from 94 surgically resected colorectal primary tumors were analyzed for EGFR expression using immunohistochemistry along with 49 paired positive metastatic nodes and 19 distant metastases. Proportional odds regression and log-rank tests described the association of tumor stage and survival with EGFR, dichotomized to low expression (0, 1) versus high expression (2, 3). High EGFR expression rates in the primary tumor, metastatic lymph node and distant metastases were 18%, 24%, and 21%, respectively. The concordance rate was 71% for stage III/IV patients. High EGFR expression in primary tumor was associated with higher tumor stage (odds ratio 4.3, P = 0.005) and thus predicted poor survival (P = 0.046). High EGFR expression in the primary tumor was not associated with survival in patients with stage III or IV disease, whereas high EGFR expression in metastatic lymph nodes was associated with relatively poor survival (P = 0.005), for both stage III (P = 0.002) and stage IV patients (P = 0.014). Concordant high EGFR expression in primary tumor and lymph nodes conferred added risk of death (P = 0.003), conversely, concordant EGFR-negative primary tumor and lymph node was protective (P = 0.097). EGFR expression may be discordant among primary, nodes, and metastases in colorectal cancer. High EGFR expressions in metastatic lymph nodes seem to be more accurate in predicting survival than in primary or metastatic tissues.

Simultaneous Assessment of Cyclin D1 and Epidermal Growth Factor Receptor Gene Copy Number for Prognostic Factor in Oral Squamous Cell Carcinomas

Cyclin D1 gene (CCND1) numerical aberrations are independent prognostic indicators of head and neck squamous cell carcinomas (HNSCCs). High epidermal growth factor receptor gene (EGFR) copy number is associated with poor prognosis in lung cancer, but such findings are controversial in oral SCCs (OSCCs). We analyzed copy number status in CCND1 and EGFR in OSCC patients and its association with clinical outcome.EGFR and CCND1 statuses were analyzed in 85 OSCC patients by fluorescence in situ hybridization (FISH) of specimens obtained by fine-needle aspiration biopsy.CCND1 numerical aberration was found in 35 of 85 tumors (41%), and aberrant EGFR copy number was observed in 36 (42%). Gene amplification (GA) was dominant among CCND1 copy number changes (14/35:40%). Balanced trisomy (BT) was the most frequently observed EGFR aberration (17/36:47%). In a multivariate Cox's proportional hazards analysis, CCND1 GA was correlated with disease-free survival (P < 0.001), whereas EGFR BT was significantly correlated with overall survival (P = 0.001). Patients with a combination of CCND1 GA and/or EGFR BT had significantly poorer clinical outcome.CCND1 and EGFR copy number changes were frequent in OSCC and had differing aberration patterns. CCND1 GA and EGFR BT statuses by dual-color FISH were the predominant predictors of clinical outcome. Further investigation is needed to determine the implications for EGFR inhibitor therapy in OSCC.

Molecular Imaging and Quantitative Measurement of Epidermal Growth Factor Receptor Expression in Live Cancer Cells Using Immunolabeled Gold Nanoparticles

The goal of this study was to assess whether immunolabeled nanoparticle biomarkers are comparable to fluorescent marker imaging in measuring epidermal growth factor receptor (EGFR) expression. EGFR expression was quantified using both imaging methods in four cell lines: A431 human epidermoid carcinoma cells, which are known to have high EGFR expression; two cell lines with lower EGFR expression (270-GBM human glioblastoma xenograft cells and H2224 human glioblastoma xenograft cells); and MDA-MB-453 breast carcinoma cells, which do not express EGFR. To enhance contrast of the nanoparticle biomarkers, a darkfield microspectroscopy system was used that includes a custom epi-illumination light train. Nanoparticle-bound cells were clearly distinguished from control cells not bound to nanoparticles in that they showed a significant increase in detected intensity under darkfield illumination due to nanoparticle scattering. The average nanoparticle-scattering intensity for A431 cells was 41.5 counts per cell compared with 24.7 for 270-GBM cells, 8.77 for H2224 cells, and 0.44 for MDA-MB-453 cells. The average fluorescence intensity for A431 cells was 35.3 counts per cell compared with 28.7 for 270-GBM cells, 5.91 for H2224 cells, and 2.07 for MDA-MB-453 cells. A plot of fluorescence intensity versus nanoparticle-scattering intensity for all four cell lines showed that the data agree with a linear relationship given by the following equation: NP = 1.0691 x FL - 0.3873, where NP is the nanoparticle-scattering intensity and FL is the fluorescence intensity. The covariance of the data with the trend line was R(2) = 0.9409. The average peak wavelength of nanoparticle scattering was 570.93 nm for A431 cells, 565.26 nm for 270-GBM cells, and 562.70 nm for H2224 cells (with no clear peaks observed for MDA-MB-453 cells). This spectral trend shows that nanoparticle scattering may reveal additional information about their nanoenvironment via refractive index sensitivity. Immunolabeled nanoparticles can quantify receptor expression with performance comparable to fluorescence markers and show promise to better characterize receptor expression via their refractive index sensitivity.

EGFR protein overexpression and gene copy number increases in oral tongue squamous cell carcinoma

New promising therapeutic agents targeting epidermal growth factor receptor (EGFR) have been developed although clinical information concerning EGFR status in oral tongue squamous cell carcinoma (OTSCC) is limited. We investigated EGFR protein expression and gene copy numbers in 78 pretreatment OTSCC paraffin samples. EGFR protein expression was found in all 78 tumours, of which 72% showed an intense staining. Fifty-four percent of the tumours had high (⩾four gene copies) EGFR gene copy numbers. EGFR gene copy number was significantly associated with EGFR protein expression ( P = 0.002). Pretreatment EGFR staining intensity tended to be associated with non-pathological complete remission after preoperative radiotherapy for Stage II OTSCC. No correlation was found between EGFR status and survival. EGFR FISH results were significantly ( P = 0.003) higher in more advanced tumours (Stages II, III and IV) than in the tumours in Stage I. Non-smokers exhibited a significantly higher EGFR gene copy number and protein overexpression in Stages I and II OTSCC than smokers ( P = 0.001, P = 0.009). In conclusion, EGFR was found to be overexpressed in all OTSCCs making this cancer type interesting for exploring new therapeutic agents targeting the EGFR receptor.

Impact of cellular folate status and epidermal growth factor receptor expression on BCRP/ABCG2-mediated resistance to gefitinib and erlotinib

The effect of folate status on breast cancer resistance protein (BCRP)-mediated drug resistance to epidermal growth factor receptor (EGFR)-targeted drugs, such as gefitinib and erlotinib, was investigated in two human colon cancer cell lines, WiDr and Caco-2, of which the latter displayed greater sensitivity to these drugs due to high EGFR expression. Caco-2 LF/LV cells, growing under low-folate (LF) conditions, showed increased BCRP protein expression compared with the high-folate (HF) counterpart, which was associated with 1.8-fold resistance to gefitinib. Of note, the BCRP-specific inhibitor Ko143 completely reverted this phenotype. WiDr LF cells also showed slightly increased BCRP expression compared with the HF cells, but no differences in gefitinib sensitivity were observed. Both Caco-2 LF/LV and WiDr LF cells showed 2.4- and 2.3-fold resistance to erlotinib, respectively, compared with their HF counterparts, which mechanistically seemed BCRP unrelated, as Ko143 had no effect on erlotinib activity. In conclusion, our data suggest that in EGFR-expressing Caco-2 cells, BCRP is one of the determinants of gefitinib resistance but not of erlotinib resistance. Beyond this, folate depletion can provoke an additional decrease in gefitinib and erlotinib activity by mechanisms dependent or independent of BCRP modulation.

Molecular determinants of response to matuzumab in combination with paclitaxel for patients with advanced non-small cell lung cancer.

Antibodies targeting epidermal growth factor receptor (EGFR) have proven to be effective in patients with non-small cell lung cancer (NSCLC) that express EGFR. We recently published a phase I study of weekly matuzumab plus paclitaxel. This therapy was well tolerated and showed clinical responses in the majority of patients. Although matuzumab displays potent antitumor activity in some patients, not all patients respond well to treatment. Whether dysregulation of EGFR-mediated pathways precludes or sensitizes cells to paclitaxel is unknown. We sought to determine molecular predictive factors for therapy response in a phase I/II study patient cohort treated with matuzumab+/-paclitaxel. Twenty-three cases [including one complete response (CR), three partial responses (PR), 10 stable diseases (SD)] were screened using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), PCR/sequencing and denaturing wave high performance liquid chromatography (D-HPLC) for expression, amplification, and mutation status of EGFR and downstream signaling pathways. All patients with PR or CR displayed an either high overall or single-cell EGFR expression in the majority of cells. In addition, all of the moderate responders, who achieved SD after at least two cycles of therapy, showed diffuse EGFR expression rates and/or strong single-cell EGFR expression. In contrast, 44% of the nonresponders showed low overall or single-cell EGFR expression levels. No low-expressing EGFR cases were present within the responder group. In addition, among patients with a gain-of-function mutation in KRAS primary therapy failure and/or short responses to therapy were observed. Our data suggest that EGFR expression and KRAS mutation status is predictive for clinical response to matuzumab +/- paclitaxel in patients with advanced NSCLC.